Method for discovering neurogenic agents

ABSTRACT

A method for discovering neurogenic drugs is revealed. The method allows for systematic screening of test agents such as libraries of compounds. The method consists of exposing test agents to cultures of differentiating neural progenitor cells and measuring their effects on increasing the overall cell number and/or the number of neurons.

[0001] The present patent application claims the benefit of U.S.Provisional Patent Application 60/432,359, filed Dec. 9, 2002, and U.S.Provisional Patent Application 60/493,674, filed Aug. 8, 2003, which arehereby incorporated by reference in their entirety and relied upon.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] According to a long-held doctrine, no significant number ofneurons are made and contribute to function in the adult mammaliannervous system. However, recent data indicate that adult mammalianbrains contain neural precursor cells capable of generating new neuronsboth in normal and in injured conditions. These new neurons have beenquantified in live animals by injecting or feeding in drinking water amarker of dividing cells, bromodeoxyuridine (BrdU) and by immunostainingof post-mortem brains with antibodies against BrdU and neuronal markers.An endogenous marker of dividing cells, ki67 protein, has also been usedinstead of BrdU for this purpose. Thus, in healthy, young rodents,approximately 3,000-15,000 new cells per day are estimated to be born inthe dentate gyrus of the hippocampus, about 60% of which express earlyneuron-specific proteins such as double-cortin and type IIIbeta-tubulin. Significant number of new cells and new neurons have alsobeen observed in healthy, young primates. In rodents as well as inprimates, the location of neurogenic areas in the CNS is limited to thedentate gyrus of the hippocampus and the subependymal layer of thestriatum. In human patients of different ages who have been diagnosedwith a tumor of the tongue, a single injection of BrdU has revealedsignificant number of new cells and new neurons being born in thedentate gyrus and the subependymal layer of the striatum. Thus, theprocess of generating new neurons (neurogenesis) occurs in the mature,adult brain in significant quantities throughout rodents, primates, andhuman species.

[0004] Such significant quantities of new neurons suggest that they maybe important for the normal physiology of the brain, especially thehippocampus. Hippocampus is the well-known center of learning, memory,and other cognitive functions, processes which new information areadded, edited, stored, and recalled constantly throughout life. Sincehippocampus is also the most potent neurogenic area of the brain, manystudies have been undertaken to establish whether neurogenesis may bethe cellular mechanism to structurally accommodate the ever-increasingvolume of cognitive processing to be handled. Thus, it has been shownthat at least some of the newly born neurons, marked by genetic markers,do mature to be electrophysiologically active and integrate into theexisting neuronal circuitry of the hippocampus. Ablation of theneurogenesis in rats leads to decreased cognitive capabilities inseveral behavior tests. Thus, the existing data demonstrate thatneurogenesis significantly contributes to the normal hippocampalphysiology.

[0005] In abnormal conditions, such as when an injury to a brain areahas occurred, neurogenesis becomes more wide-spread and perhapsfunctionally diverse. In rodent models of ischemic and hemorrhagicstroke, the newly born neurons of the subependyma (also referred to assubventricular zone) are seen migrating to and accumulating in thelesion area of the cortex. However, the newly born neurons have shortsurvival period.

[0006] Thus, a compound that can stimulate the endogenous neurogenesiseither in a disease state or in a healthy state may be an effective drugfor a number of human nervous system diseases. However, the currentlimitation is the lack of an effective, predictive in vitro assay thatcan be used to select a neurogenic compound for clinical drugdevelopment. Disclosed here is a novel, in vitro assay, which iseffective and predictive, to be useful for discovering a compound thatpromotes neurogenesis in vivo. Also disclosed are classes of compoundstructures that are shown to be particularly effective in promoting theneurogenesis.

[0007] This invention relates to the method of discovering a neurogenicdrug to treat neurologic, psychiatric, and aging-related disorders. Italso relates to the use of Fused Imidazoles, Aminopyrimidines,Nicotinamides, Aminomethyl Phenoxypiperidines and Aryloxypiperidines foruse as therapeutic agents and analytical reagents by means of promotingneurogenesis. More particularly this invention relates to these agentsas therapeutics for prevention and treatment of neurological diseases inmammals and reagents for detecting neurogenesis and proliferation.

[0008] 2. Description of the Related Art

[0009] Most antidepressants are thought to work by increasing the levelsof monoamines available for post-synaptic receptors. Examples of classesof agents working apparently by the “monoaminergic hypothesis ofdepression” include the selective serotonin uptake inhibitors (SSRIs)like fluoxetine, the mixed noradrenaline/serotonin transporter blockerslike tricyclic agent imipramine and noradrenaline uptake inhibitors likedesipramine. The antidepressant-induced increase in intraneuronalbiogenic amines occurs quite rapidly. However, theantidepressant-induced improvement in clinical behavior requires weeksof daily administration.

[0010] One hypothesis that may account for the slow-onset of theantidepressants' therapeutic activity is that they work by promotinghippocampal neurogenesis. It is expected that neurogenesis would requirea number of weeks for stem cells to divide, differentiate, migrate andestablish connections with post-synaptic neurons. The neurogenesistheory of depression then postulates that antidepressant effect isbrought about by structural changes in the hippocampal circuitrycontributed by newly generated neurons stimulated by antidepressants(Malberg et al., 2000; Czeh et al, 2001; Santarelli et al, 2003).

[0011] The neurogenic theory of depression, though not conclusive, hasstrong supportive data including the finding that neurogenesis isactually requisite for antidepressant behavioral improvement in thenovelty suppressed feeding model (Santarelli et al., 2003). Atherapeutic benefit from hippocampal neurogenesis is further supportedby the finding of hippocampal atrophy in depression, where MRI imagingstudies identified a reduction in the right and the left hippocampalvolumes in individuals with major depression (Sheline et al., 1996;Bremner et al., 2000; Mervaala et al., 2000). Long standing works alsosuggest a strong relationship between glucocorticoid dysregulation orglucocorticoid hypersecretion in stress and depression, such that thehippocampal volume loss might be considered a consequence ofglucocorticoid-induced hippocampal neuronal loss (Sheline et al., 1996;Lucassen et al., 2001; Lee et al., 2002 (review)). Furthermore, instudies which involved the administration of a chronic stress toanimals, both hippocampal volume changes and reduction in neurogenesiswere observed, and these events were both reversed by chronicantidepressant administration (Czeh et al., 2001; Pham et al., 2003),further illustrating the strong association between depression, stressand neurogenesis. The association comes full circle, since agents orconditions that promote a reduction in neurogenesis also appear ascausative agents/events in depression, specifically glucocorticoid(Sapolsky, 2000), and depletion of serotonin (Brezun and Daszuta, 1999).Kempermann and Kronenberg (2003), though acknowledging the validity ofthe hippocampal neurogenesis theory of depression, suggest that thishypothesis needs to be looked at in the context of a larger model ofcellular plasticity, which elucidates how antidepressants induce nascentneurons of unknown phenotype to survive and produce viable circuits.

[0012] Neurogenesis can be characterized as three successive stages:proliferation of endogenous stem cells and precursors, differentiationinto neurons and neuron maturation with formation of viable synapticconnections (plasticity). By taking into account all stages ofneurogenesis, then the hippocampal volume loss in depression couldpotentially be caused by 1) inhibition of the endogenous hippocampalstem cell proliferation in the dentate gyrus, 2) inhibition ofdifferentiation and dendrite development and 3) by loss of neurons(apoptosis) and their dendritic structure. Though apoptosis is observedin depression, hippocampal apoptosis as measured by DNA fragmentationfrom depressed patients appears to play only a minor role in the volumeloss (Lucassen et al., 2001). In an animal model of acute stress or innormal animals receiving exogenous corticosterone the stress did cause areduction in synaptic plasticity in the hippocampus (Xu et al., 1998).Chronic administration of the tricyclic antidepressant, imipraminepartially reversed the loss in LTP in socially stressed, depressive-likeanimals (Von Frijtag et al., 2001) suggesting imipramine effects on theplasticity phase of neurogenesis. In another animal model of depression,presenting neurogenesis loss and hippocampal volume loss, stressedanimals that chronically received the antidepressant, tianeptine, showedsimilar numbers of dividing cells as control animals (no social stress)a measure of proliferation (Czeh et al., 2001). In an experiment lookingat association of antidepressants and neurogenesis in normal adult rats,the antidepressant, fluoxetine required chronic administration to causeproliferation of cells in dentate gyrus (24 hrs post treatment), butthere was considerable loss of nascent cells whether in the presence orabsence of fluoxetine treatment, where fluoxetine provided no observeddifferentiation or survival benefit (Malberg et al., 2000). Results ondifferent neurogenic intervention points by known antidepressantssuggest that novel neurogenic agents that intervene at different pointsin the neurogenesis pathway, could result in potentially diversetherapeutic effects on depression.

[0013] These points of intervention can be studied and the targetelucidated for novel antidepressant candidates through in vitro assays.Since adult stem cell proliferation and neurogenesis is observed inadult vertebrates in hippocampal dentate gyrus (Gould et al., 2001;Eriksson et al., 1998), we can use multi-potential hippocampal stemcells to screen agents in vitro for neurogenic activity.

[0014] Interestingly, chronic administration of either theantidepressant fluoxetine, an SSRI or the antidepressant rolipram, aphosphodiesterase IV inhibitor, promoted neurogenesis in normal animals(Malberg et al., 2000; Nakagawa et al., 2002). One might conclude fromthese results that any agent that promotes neurogenesis will generate abehavioral benefit in depression, unrelated to the agentsmechanism-of-action or possibly there is a common pathway where bothdrug actions overlap. D'Sa and Duman (2002) suggest a scheme whereby thephosphorylation and activation of CREB and the subsequent expression ofBDNF are central to the induction of neurogenesis, that culminates inantidepressant behavior. CREB phosphorylation is increased in animalsadministered rolipram chronically (Nakagawa et al., 2002) andantidepressants that either increase Ca2+/CaM-kinases or cAMP couldcause the phosphorylation of CREB in the nucleus (reviewed by D'sa andDuman 2002). They further suggest that the phosphorylated CREB thenbinds to CRE binding site to promote the expression of BDNF and bcl-2,that appear critical to cell survival and plasticity. Proof ofneurotrophic factor BDNF's involvement in depression comes from studiesshowing that antidepressants and electroconvulsive shock both caused anincrease in BDNF levels (Nibuya et al., 1996) and that intrahippocampalinjection of BDNF had antidepressant activity in two models ofdepression (Shirayama et al., 2002).

[0015] If neurogenesis is critical for antidepressant activity is italso sufficient and is the mechanism by which the neurogenesis occurs ortiming of neurogenesis also critical to the therapeutic activity? We cantry to answer these questions using novel agents developed throughscreening paradigms that identify agents that promote the proliferationand differentiation of endogenous hippocampal stem cells to neurons invivo if they will be effective antidepressants. Since we have completedthe screening of 10,000 small molecule compounds in in vitro models ofneurogenesis and shown that our in vitro screen is predictive of in vivoneurogenic efficacy, we can then test these orally available agents,that promote in vivo neurogenesis, in models of depression. Rolipram, anantidepressant that works by increasing cAMP levels and is neurogenic inanimals (Nakagawa et al., 2002) was effective in our primary in vitroneurogenesis screen. This suggests that our primary in vitro screenwould include those agents that might promote neurogenesis by targetingthe cAMP/pCREB/BDNF pathway. This does not necessarily exclude all otherneurogenesis mechanisms for our NSI compounds. If the target of theseneurogenic agents are important for behavioral activity where threeseparate chemically diverse classes showed in vitro assay efficacydifferences and that the mechanism for all does not overlap at the pointof CREB phosphorylation and BDNF expression then we might expect verydifferent effects on behavioral activities in depression models.

[0016] Neuropathology associated with key cognitive regions of theAlzheimer's diseased brain have led to therapeutic strategies thataddress the neuronal loss, in the hopes of reducing the cognitivedecline. One strategy enlists trophic agents, that regulate neuronalfunction and survival, as AD therapeutics (see Peterson and Gage, 1999).Problems with systemic administration, side effects and locatingtrophic-sensitive neurons generated few clinical successes with thesetherapies. One AD therapeutic, AIT-082, promotes memory enhancement inAD individuals potentially by stimulating endogenous trophic factors(Ritzman and Glasky, 1999; Rathbone et al., 1999). So the use of agentsto promote increased survival and function of the remaining availableneurons appears to have some therapeutic value.

[0017] Hippocampus is one of the main brain regions where neurogenesisin adult brain has been documented across several vertebrate species,including monkeys and humans (e.g., Gould et al., 2001; Eriksson et al.,1998). In fact, adult hippocampal neurogenesis contributes functionallyto cognitive capacity. Shors et al. (2001) reported that inhibition ofneurogenesis in adult rat hippocampus, in the absence of the destructionof existing neurons, caused impaired memory function. Many studiesobserved that degenerative conditions induced neurogenesis in maturemammalian brains, suggesting the existence of a natural repair pathwayby means of neurogenesis. A focal ischemic model, reversiblephotothrombic ring stroke, caused increased neurogenesis in rat cortexby 3-6% (Gu et al., 2000). Seizures induced by electroconvulsive shockin adult rats increased neurogenesis in dentate gyrus of hippocampus(Scott et al, 2000; Madsen et al, 2000). Also, rats gamma-irradiated tokill mitotic cells in the CNS showed reduced numbers of nascent neuronsand reduced LTP in slice cultures. These observations highlight thelikelihood that a cellular mechanism for neurogenesis within adult humanCNS, especially in hippocampus, does exist both as a normalphysiological process and as a self-repairing pathway.

[0018] In adult neurogenesis a decline due to aging is observed (Kuhn etal., 1996), though proof that this age-dependent decline in neurogenesiscauses cognitive impairment is still debated. Considerable evidence doesexist, indicating that increased neurogenesis reduces age-associatedcognitive decline. This is most dramatically observed with thetransplantation of human stem cells into aged rats initiating improvedwater maze learning and retention (Qu et al., 2001). Other data suggeststhat induction of neurogenesis by diet restriction (Lee et al., 2000)exercise (van Praag et al., 1999) or growth factor addition(Lichtenwalner et al., 2001) improves learning and memory in adult oraged rats. A number of other inducers of neurogenesis have beenidentified, including anti-depressants (Malberg et al., 2000; Czeh etal., 2001), and nitric oxide donors (Zhang et al., 2001) suggesting theusefulness of neurogenic agents for other diseases presentingcognitive-deficits, such as stroke and depression. A small molecule thatinduces hippocampal neurogenesis that is blood brain barrier penetrablewould allow for a potentially novel oral therapeutic for Alzheimer'sdisease.

[0019] Other potential AD therapeutics progressing in clinical trials,target neurodegeneration in the hopes of reducing the neuronal loss andcognitive decline. Apoptotic death involving caspase pathways and DNAfragmentation has been measured in in vitro and animal models of AD andin Alzheimer's diseased brain tissue. The extent of apoptosis leading toneuronal loss is of continual debate with most agreeing it has someeffect, but that other neuronal death pathways definitely play a role(see Behl, 2000; Broe et al., 2001; Roth, 2001). Concern that measuresof upstream caspase markers in neurons from AD tissue may not proceed todegeneration has been suggested (Raina et al, 2001). In order to screenfor a neuroprotectant therapeutics it is critical, therefore, to measuremore than one endpoint of neuronal death and determine at what point anagent may intervene in the death pathway(s). Behl (2000) suggested thatAD pathology is most likely a mixture of apoptotic and necrotic pathwaysand that concentrating therapeutic discovery using only one pathway mayprovide inconclusive results. All hits in our neurogenesis models weretested through our secondary apoptosis/necrosis assay to screen foragents that function both as neurogenic and neuroprotective agents.These agents may improve or reverse the cognitive decline observed inMCI and AD.

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[0083] Xu, L., Holscher, C., Anwyl, R., Rowan, M. J. (1998).Glucocorticoid receptor and protein/RNA synthesis-dependent mechanismsunderlie the control of synaptic plasticity by stress. PNAS USA. vol95:3204-3208.

[0084] Zhang, R., Zhang, L., Zhang, Z., Wang, Y., Lu, M., Lapointe, M.,and Chopp, M. (2001) A nitric oxide donor induces neurogenesis andreduces functional deficits after stroke in rats. Ann. Neurol. vol. 50(5), pp 602-11.

SUMMARY OF THE INVENTION

[0085] A neurogenic drug is an agent that enhances the process ofgenerating new neurons (neurogenesis). Recent studies indicate thatneurogenesis occurs in the adult human brains under normal as well asunder degenerative conditions and that such adult-generated neurons docontribute functionally to the brain physiology such as learning andmemory. These observations highlight the likelihood that a cellularmechanism for neurogenesis within adult human CNS, especially inhippocampus, does exist both as a normal physiological pathway and as aself-repairing pathway. What is lacking and contributes to permanentdamage may be (1) the volume/persistence of neurogenesis and/or (2) thesurvival/maturation of the new neurons. The objective of theneurogenesis screen as described here is to discover a compound thatwill significantly boost either of these processes.

[0086] Many neurological diseases, including Alzheimer's disease, mildcognitive impairment, dementia, age-related cognitive decline, stroke,traumatic brain injury, spinal cord injury and the like areneurodegenerative conditions. Neuropsychiatric diseases includingdepression, anxiety, schizophrenia and the like also show nerve celldysfunction leading to cognitive, behavioral, and mood disorders. Aneurogenic drug would be beneficial for countering and treating thesediseases.

[0087] The present invention discloses a method of discovering such aneurogenic drug. Such drug will serve to prevent or treatneurodegenerative and neuropsychiatric disorders by promoting the birthof new neuron endogenously within the nervous system by administeringthe compounds of the present invention into the patient. This mayinvolve delivery of the agents alone or together with transplanted stemcells or progenitor cells.

[0088] Using the method herein, compounds of the type, Fused Imidazoles,Aminopyrimidines, Nicotinamides, Aminomethyl Phenoxypiperidines andAryloxypiperidines are evaluated for their ability to promoteneurogenesis by proliferation/differentiation of human hippocampalmultipotent stem/progenitor cells and neuronal progenitors.

BRIEF DESCRIPTION OF THE DRAWINGS

[0089]FIG. 1. Schematic description of neurogenesis processes capturedin the assay and different potential sites of a neurogenic drug action.

[0090]FIG. 2. Detection of changes in cell number by Alamar Blue dye.Alamar Blue, a fluorescent dye, is used as an indicator of metabolicrespiration to determine optimum plating density. Results at an initialplating density of 30,000 cells/well suggest a large difference in cellnumber on removal of mitogen from the N2b media (differentiation) versusN2b with mitogen (proliferation) conditions. This figure only describestotal cellular activity, further markers are required to determine whatcell types (e.g. neuronal, glial) are observed under differentiatingmedia.

[0091]FIG. 3A. Influence of known growth factors on proliferation andneurogenesis relative to control. Hippocampal progenitor cells weretreated for seven days with differentiation media (without mitogen) inthe presence or absence of 20 ng/ml of growth factor dosed every otherday. Plates were treated with Alamar Blue as described in Methods, thenfixed and stained with antibody (TuJ1) against type III beta-tubulin(neuronal marker). The 96-well plate was read in a fluorescent platereader. Bars represent the Mean+SD from 4 wells per treatment.

[0092]FIG. 3B. LIF effects on hippocampal cell proliferation andneurogenesis by manual cell counting. Hippocampal progenitor cells weretreated for seven days with differentiation media (without mitogen) inthe presence or absence of 20 ng/ml LIF. Three fields were analyzed perwell for total number of cells (DAPI positive nuclei) and for totalnumber of neurons (TUJI positive cells). Bars represent the Mean+SD from4 wells per treatment. The percentage of neurons calculated for eachtreatment are as follows: 48.5+6.3% for controls and 53.6+1.15 for LIF.The non-TUJ1 positive cells are mainly astrocytic (GFAP+).

[0093]FIG. 4. Examples of proliferation profile of compounds selectedfrom primary screening. Proliferation was measured after compoundtreatment for 7 days by Alamar Blue staining of live cells per well.Shown are relative values over the vehicle control.

[0094]FIG. 5. Example of neurogenesis profile of compounds selected fromprimary screening. After 7 days of compound treatment, the ratio ofneuron number (TuJ1 stained) to the total nuclei number (Hoechststained) was determined. Shown are the relative ratio of neuron:totalcells for each compound over the vehicle control in percentage. Typicalratio for vehicle control is 40-50% neurons. The ratio can change byeither increased differentiation of the cells to neurons, decreasedproliferation of astrocytes, or increased proliferation of neuronalprogenitors.

[0095]FIG. 6. Examples of neurogenesis profile of compounds selectedfrom primary screening. After 7 days of compound treatment, the cellswere stained with TuJ1 for neurons. The absolute number of TuJ1+ neuronsper area was quantified and expressed as a relative value to the vehicletreated control.

[0096]FIG. 7. Dose-dependent increase in neuron number. Differentiatinghuman hippocampal progenitor cells were treated for 7 days with varyingconcentrations of “primary hits”. Subsequently, the cells were fixed,stained with TuJ1, and positive cells were quantified by an automatedcell counter. Shown are the number of neurons after each treatmentnormalized against the vehicle control (0 microM=1.0).

DETAILED DESCRIPTION OF THE INVENTION 1. A Stable Cell Line of NeuralProgenitors

[0097] A screening of a large number of unknown agents (e.g., proteinfactors, peptides, nucleic acids, natural compounds, or syntheticcompounds) for discovering a candidate drug involves repeating the sametest for several hundreds to several million times. This requires agreat deal of reproducibility from the test. In order to obtain suchreproducibility for neurogenesis assay, we have created stable celllines of neural progenitors, which upon differentiation generatereproducible quantities of neurons. In a preferred embodiment, amultipotent neural stem/progenitor cell line derived from humanhippocampus was used. Cell lines derived from other CNS areas, includingdentate gyrus of an adult brain, can also substitute. A neuralprogenitor population derived as a stable cell line from partialdifferentiation of embryonic stem cells can also be used. For thispurpose, a cell line is defined as a population of cells having beenexpanded for at least 10 cell-doublings.

[0098] Cell lines that are genetically engineered to enhance the cells'mitotic capacity can also be used. In a preferred embodiment, thegenetic modification consists of over-expression of functional c-mycprotein intracellularly under a conditional activation system such asc-myc protein fused to a ligand-binding domain of an estrogen receptor.Cell lines that are not genetically engineered are preferred and canalso be used.

[0099] In a preferred embodiment, a progenitor population that upondifferentiation generates both neurons and glia in a single culture hasbeen used. Presence of glia, either astrocytes and/or oligodendrocytesor their precursors, are required to promote physiological maturation ofnascent neurons born from their precursors in culture.

[0100] In a preferred embodiment, differentiation of the progenitors isinitiated by withdrawing the mitogen from the culture. Serum as well asother growth-promoting factors should be avoided from thedifferentiating culture since they will significantly affect thereproducibility and interfere with the neurogenesis assay.

2. Preparation of Assay Plate

[0101] Neural stem/progenitor cells differentiate spontaneously in theabsence of a mitogen. Undifferentiated mitotic cells are harvested byenzyme treatment to remove residual mitogen, in the preferredembodiment, basic fibroblast growth factor (bFGF). The collected cellsare seeded into appropriate plates (standard 96-well or 384-well)pre-coated with the usual extra cellular matrix proteins (poly-D-lysineand fibronectin, for example) for attachment of the cells. The initialseeding density can be within the range of about 2,000-125,000 cells perwell of a 96-well plate. The preferred density is 40,000 cells per wellof a 96-well plate, which has been optimized for best signal-to-noiseratio. Too low cell density retards the initiation of differentiationand results in poor plating efficiency, which interferes with the assay.Too high cell density leads to inhibition of neurogenesis due tocell-cell contact and paracrine factors, which also interferes with theassay. The actual cell number can be proportionally decreased orincreased depending upon the surface area of the culture substrate used.For example, for a 384-well plate, which has approximately ¼ of thesurface area of a 96-well plate, the initial seeding density should bedecreased accordingly (¼).

3. Detection of Neurogenesis

[0102] The key activity of a neurogenic drug is to increase the numberof neurons generated from their precursors. A molecule can bring aboutsuch increase in the neurogenesis by a number of different mechanisms.It can act as a mitogen for the neural stem/progenitor cells andincrease the progenitor's cell number, which in turn results inincreased number of neurons in the culture when differentiated. Or, itcan act as a neuronal specification factor by promoting thestem/progenitor cell differentiation toward neurons in the expense ofglia. This will also result in increased number of neurons in theculture, but without changing the overall cell number. Or, it can act asa mitogen for committed neuronal progenitors that differentiate onlyinto neurons. Increasing this subpopulation would also increase thefinal number of neurons in the culture. Or, it can act as a survivalfactor to rescue immature neurons from undergoing cell death duringdifferentiation, which will result in increased neurons (FIG. 1).

[0103] The assay method here captures all of these possibilities byallowing for sufficient time for these processes to unfold. In apreferred embodiment, for human neural stem/progenitor cells, the assayis continued for seven days. A minimum of three days from the onset ofdifferentiation should be allowed for stable expression of definitiveneuronal markers to appear. A sufficient time is also required for acompound action on differentiation and/or proliferation to take place toa sufficient degree to be reliably detectable. Manifestation ofdrug-induced changes in neuron number takes a minimum of three days forthe human cells to be detectable.

[0104] The final neuron number is detected by immunostaining of theculture with antibodies against neurons and quantified by counting ofthe immunopositive neurons and/or by measuring the staining intensity.

4. Method for Measuring Neurogenesis

[0105] (1) Undifferentiated human neural stem/progenitor cells wereharvested by enzyme treatment.

[0106] (2) The collected cells were seeded at 40,000 cells per well of96-well plates pre-coated with extracellular matrix proteins (e.g.,Biocoat PDL, Fisher). The seeding media is a standard serum-free, growthfactor-free, basal media that supports healthy neuronal/glial survival,such as N2 without phenol red.

[0107] (3) Test agents at appropriate concentrations were added to eachwell on Day 0.

[0108] (4) The assay plates were incubated for 7 days, with 50% mediachange at every other day. On Day 2, 4, and 6 of post-plating,additional increment of the screening agents at appropriateconcentrations were added to each well.

[0109] (5) On the final day of the culture (Day 7), alamar blue dye wasadded to each well and the cultures were further incubated for 2 hoursat 37° C.

[0110] (6) The fluorescence of the oxidized dye in each well was read bya fluorescent plate reader with the following settings:

[0111] Read Mode End Point

[0112] Excitation 530 nm, emission 590 nm, cutoff 570 nm

[0113] The fluorescence level is proportional to the number of respiringcells in the culture and is a measure of a proliferative activity of atest agent (FIG. 2).

[0114] (7) After the alamar blue assay, the cells were fixed and stainedwith antibodies against neuron-specific antigens according to standardprocedures. Typical antigens effective were TypeIII-beta tubulin andMAP2c.

[0115] (8) The total cell number in each well was quantified by stainingthe cultures with a nuclear dye such as DAPI or Hoechst according tostandard procedures.

[0116] (9) As a preliminary detection of positive activities, theoverall immunostaining intensity in each well was read by a fluorescenceplate reader. For the positive hits, more quantitative analysis wascarried out by automated morphometric counting of individual cells.

5. EXAMPLES Example 1

[0117] Selection of a positive control.

[0118] Several neurotrophic factors—including brain-derived neurotrophicfactor, glia-derived neurotrophic factor, neurotrophic factor-3, andleukemia inhibitory factor—suggested to have neurogenic properties weretested in the assay described above. Only one (leukemia inhibitoryfactor) was effective (FIGS. 3A and 3B). Thus, the assay candiscriminate test agents for selectively having a neurogenic activity.The positive control utilized is leukemia inhibitory factor (LIF), acytokine growth factor, at 20 ng/ml. The selection of LIF as thepositive control is based on its properties to increase by 2-3 fold thenumber of neurons and glia. This effect validates both the neural stemcell system, in which, should a compound be effective in neurogenesis,the cells respond appropriately by enhanced differentiation and/ormitosis, and the assay method in which such cellular responses can bemeasured reproducibly and quantifiably.

Example 2

[0119] Primary screening of unknown compounds.

[0120] 5,628 synthetic compounds of the type Fused Imidazoles,Aminopyrimidines, Nicotinamides, Aminomethyl Phenoxypiperidines andAryloxypiperidines are evaluated for their effect on neurogenesisaccording the assay method described above. From the preliminaryanalysis using the fluorescent plate reader, over 300 compounds to dateshowed initial positive activity. Those were re-analyzed by quantitativeneuron counting. Among them, 30 compounds significantly increased cellnumber (“proliferation”, FIG. 4); 53 increased the number of neurons(“neurogenesis”, FIG. 5 & FIG. 6); and 7 showed significant activity inboth. The significance level was empirically set at an activity above30% change over the vehicle control for proliferation and above 10%change for neurogenesis. A summary of the result in the compoundscreening is provided in Table I. TABLE I Summary of Compound ScreeningPrimary Hits Proliferation Neurogenesis Double Screen Confirmed Hit HitHit 0 0 0 0 0 2,240 88 13 8 1 5,628 >300 30 53 7

Example 3

[0121] Dose-response profiles.

[0122] Linearity of dose-response and in vitro neurotoxicity are used tofurther filter down desired compounds from the primary screen. Thedose-response curve measures neurogenesis over a concentration range of100 picaM to 100 microM. The rationale for this is to eliminate early onthose compounds with pronounced toxicity and those without adose-dependent effect on neurogenesis. Examples of several primary hitsfully analyzed for dose-response are shown in FIG. 7. Significantly,most compounds exhibit a linear response over several log concentrationsbelow 1 microM. This indicates that the assay for primary screening isreliable and that the quality of the compound library is high. Table IIcontains the summary of EC50 of each compound tested. On the other hand,at high concentrations (100 microM), some, but not all showed high levelof neurotoxicity, indicating that analyzing dose-response curves will bediscriminatory and serve as an effective early filter. TABLE II ActivityProfile of Primary Hits in Vitro Neuron Proliferation Ratio EC50 forOther Compound (% of (% of Neuron Characterization ID Control) ControlNumber Of Toxicity NSI-106 211 ± 48  92 ± 6 0.1 nM r² No Toxicity 0.75NSI-144 149 ± 15 137 ± 8 1.0 nM r² No Toxicity 0.54 NSI-152 174 ± 49 112± 4 0.1 nM r² Toxic At 0.84 Highest Dose NSI-154 211 ± 63 102 ± 6 0.3 nMr² No Toxicity 0.79 NSI-155 198 ± 44 118 ± 8 0.05 nM Toxic At r² 0.49Highest Dose NSI-163 208 ± 25  120 ± 11 1.0 nM r² Toxic At 0.81 HighestDose

Utilities of the Invention

[0123] In one aspect of this invention an agent would be administered totreat a neurodegenerative disease. In a preferred embodiment of thisinvention the neurodegenerative disease would be Alzheimer's disease,dementia, mild cognitive impairment, aged-related cognitive decline,Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis,demyelination, stroke, spinal injuries, traumatic injuries, neuropathicpain, and the like.

[0124] In another of its aspects, this invention the agent would beadministered to treat a psychiatric disease. In a preferred embodimentof this invention the psychiatric disease is depression, post-traumaticstress syndrome, stress, anxiety, schizophrenia, sleep deprivation,cogntive dysfunction, amnesia, and the like.

[0125] In another aspect of the invention an agent would be administeredby any number of routes and multipotent stem cells or differentiatedmultipotent stem cells would be transplanted into brain.

[0126] In another aspect of the invention the structures of the formulaare utilized in above methods:

[0127] While the invention has been described in connection with what ispresently considered to be the most practical and preferred embodiments,it is to be understood that the invention is not limited to thedisclosed embodiments, but on the contrary is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

[0128] Thus, it is to be understood that variations in the presentinvention can be made without departing from the novel aspects of thisinvention as defined in the claims.

[0129] All patents and articles cited herein are hereby incorporated byreference in their entirety and relied upon.

What is claimed is:
 1. A method of selecting a neurogenic agent,comprising the steps of: (a) obtaining a stable neural progenitor cellline; (b) plating undifferentiated neural progenitor cells into an assayplate; (c) culturing the neural progenitor cells in a serum-free,mitogen-free medium; (d) exposing the neural progenitor cells to a testagent; and (e) measuring a change in the number of neurons.
 2. Themethod of claim 1, wherein the neural progenitor cell line is derivedfrom mammalian CNS.
 3. The method of claim 1, wherein the neuralprogenitor cell line is derived from human CNS.
 4. The method of claim1, wherein the neural progenitor cell line is derived from humanhippocampus.
 5. The method of claim 1, wherein the neural progenitorcell line is derived from human subventricular zone.
 6. The method ofclaim 1, wherein the neural progenitor cell line is derived frommammalian pluripotent or totipotent stem cells.
 7. The method of claim1, wherein the neural progenitor cell line is capable of differentiatinginto neurons and glia.
 8. The method of claim 1, wherein the neuralprogenitor cell line is capable of differentiating into neurons.
 9. Themethod of claim 1, wherein the test agent is a fused imidazole, asdescribed in Structure Formula
 1. 10. The method of claim 1, wherein thetest agent is a aminopyrimidine, as described in Structure Formula 2.11. The method of claim 1, wherein the test agent is a nicotinamide, asdescribed in Structure Formula
 3. 12. The method of claim 1, wherein thetest agent is an aminomethyl phenoxypiperidine, as described inStructure Formula
 4. 13. The method of claim 1, wherein the test agentis an aryloxypiperidine, as described in Structure Formula
 5. 14. Amethod for treating neurodegenerative and neuropsychiatric disorderscomprising the step of administering a fused imidazole, as described inStructure Formula 1, to a patient in need thereof.
 15. A method fortreating neurodegenerative and neuropsychiatric disorders comprising thestep of administering an aminopyrimidine, as described in StructureFormula 2, to a patient in need thereof.
 16. A method for treatingneurodegenerative and neuropsychiatric disorders comprising the step ofadministering a nicotinamide, as described in Structure Formula 3, to apatient in need thereof.
 17. A method for treating neurodegenerative andneuropsychiatric disorders comprising the step of administering anaminomethyl phenoxypiperidine, as described in Structure Formula 4, to apatient in need thereof.
 18. A method for treating neurodegenerative andneuropsychiatric disorders comprising the step of administering anaryloxypiperidine, as described in Structure Formula 5, to a patient inneed thereof.